Wound healing is impaired by the presence of necrotic tissue, which contains dead cells and debris within second (deep partial thickness) and third degree (full thickness) burns (eschar) and chronic ulcers (e.g., diabetic ulcers). Necrotic tissue is devitalized tissue and its accumulation can result in a prolonged inflammatory response and mechanical obstruction of the wound contraction process with impeded reepithelialization, preventing wounds from healing and, if needed, hindering the wound bed from taking skin grafts. Necrotic tissue is susceptible to bacterial infection, which further impedes wound healing, and may induce sepsis in severe cases. When necrotic tissue is yellow or tan colored and a stringy mass in appearance, it is often termed slough, and when the tissue is desiccated and forms a thick and leathery texture, it is referred to as eschar. Debridement (removal) of devitalized tissue is needed to enhance wound closure. The debriding composition of this invention is thus useful in treating wounds.
Removing necrotic tissue helps to restore circulation at the wound site as adequate oxygen delivery to the wound is critical to healing. Methods to debride necrotic tissue include surgical debridement, enzymatic debridement, autolytic debridement, biological debridement, and mechanical debridement, with surgical and enzymatic debridement being most prevalent. Surgical debridement is the fastest and most efficient method for debridement and is performed by trained medical personnel/surgeons. However, viable tissue can be inadvertently removed because of a lack of clear demarcation between necrotic and viable tissue, resulting in an enlarged wound area and increased blood loss. Enzymatic debridement utilizes less training and it is generally performed by nursing/clinical staff, often requiring a longer duration of treatment for necrotic tissue removal. Enzymatic debridement involves the use of enzymes obtained from outside the body to remove non-living tissue. Debridement enzymes cleave (cut, digest, hydrolyze) large components of biological materials, particularly biomacromolecules into smaller molecules that can dissolve and be removed. Most debridement enzymes function as proteases by cleaving protein polymer chains, and a wide variety of proteases have been studied in this regard. Debridement enzymes are fast acting catalysts that produce slough of necrotic tissue. Fibrinolysin is a plasma enzyme which, after being activated, attacks fibroin components in blood clots and exudates. Deoxyribonuclease is a pancreatic enzyme that specifically attacks nucleoprotein components of purulent exudates. Trypsin and chymotrypsin are nonspecific pancreatic enzymes, but can sever protein backbones at specific amino acid residues. Enzymes from other animals include krillase, a protease derived from Antarctic krill. Tropical fruit-bearing plants provide a major source of debridement enzymes. Bromelain is a group of proteolytic enzymes from the stem of pineapple plants, which include three cysteine proteases. Papain is a nonspecific cysteine protease from papaya latex that cleaves a wide variety of substances in necrotic tissues, including fibroin, collagen, and elastin. Ficin is a nonspecific cysteine protease of similar operating pH characteristics, and is derived from a plant latex of the ficus (fig) plant. Bacteria are also a source of debridement enzymes. Subtilisins, derived from Bacillus subtilis, are mixtures of nonspecific, water-soluble serine proteases that degrade necrotic tissues. Collagenases, which are metallopeptidases, are proteolytic enzymes that degrade collagen and are derived from Clostridium histolyticum. Vibriolysin is another collagen-attacking metallopeptidase, and it is derived from the bacteria Vibrio proteolyticus. Thermolysin is a bacterial debridement enzyme from Bacillus thermoproteolyticus that acts nonspecifically with outstanding productivity, even at high temperature. Streptokinase, a fibrinogen activating protease from Streptococcus spp. and streptodomase, a deoxyribonuclease from hemolytic streptococci, has also been used in debridement (US Patent Application Number 2008/0044459).
A number of non-FDA regulated topical proteolytic enzyme products were marketed prior to 2009 for enzymatic debridement but manufacturing and distribution were stopped by FDA because of adverse allergic events and/or lack of efficacy, including papain (papaya), trypsin and chymotrypsin (pancreatic enzymes), and Bacillus subtilis proteases. Currently, there are two proteolytic enzymatic debridement products available in the United States, FDA approved Santyl® Ointment (Smith & Nephew), a collagenase derived from Clostridium histolyticum that selectively digests triple helical collagen and utilizes 250 collagenase Units per gram of white petrolatum, and Debrase® Gel Dressing (NexoBrid), a mixture of bromelain enzymes derived from Ananas comosus (pineapple). Importantly, Santyl® Ointment cannot be used in conjunction with silver and iodine antimicrobials as they deactivate the collagenase. Debrase®(MediWound Ltd.) has recently received FDA orphan drug status for eschar debridement.
U.S. Pat. No. 4,668,228 discloses a debriding tape comprising an adhesive mass on a non-gel, non-bioerodable, biocompatible occlusive or semi-occlusive backing, where an effective amount of a debriding proteolytic enzyme in dry powdered form is situated on the adhesive surface. When the powder is brought into contact with wound exudates the entire load of enzymes is released immediately.
In U.S. Pat. No. 5,206,026 is disclosed a film for instantaneous delivery of a proteolytic enzyme to a wound. When exposed to aqueous liquid the film rapidly dissolves, thus releasing its contents of enzymes simultaneously.
In U.S. Patent Application 2002/0114798 is disclosed an enzymatic wound debrider that uses a combination of a proteolytic enzyme and an anhydrous hydrophilic poloxamer carrier.
In U.S. Pat. No. 5,120,656 there is provided a process for the debridement of harvested bone having its periosteum intact, which comprises contacting the periosteum with a solution of enzyme selected from the group consisting of proteolytic collagen-digesting enzyme and mixtures thereof under enzyme activity promoting conditions to loosen the periosteum from the underlying bone surface and removing the loosened periosteum from the bone.
In U.S. Pat. No. 7,368,128, a dressing for debridement of necrotic and non-viable tissue in a wound is described, wherein the dressing comprises an effective amount of one or more proteolytic enzymes incorporated in a degradable polymeric material. The dressing of the invention provides effective debridement of necrotic wounds over a prolonged period of time, as the enzymes may be released over time.
In International Patent Publication Number WO 2012/155027, wound debridement compositions contain the proteolytic enzyme Seaprose (also known as Protease S, from the fungus Aspergillus melleus). The major enzyme in Seaprose is a semi-alkaline protease with a molecular weight around 31 kDa. It can also contain other enzymes such as amylase, a hydrolytic enzyme that breaks down carbohydrates.
In International Patent Publication Number WO1984/002846, a topical ointment for skin surface wounds is described comprising wound-healing amounts papain, bromelain, trypsin, chymotrypsin, pancreatin, lipase, amylase, aloe extract and an organic astringent agent formulated in a carrier mixture of penetrating and non-penetrating emollient oils and a polyhydric alcohol emollient, with a plurality of protease. The ointment is reported to reduce inflammation at the site of skin-surface wounds and acts to enhance the normal anti-inflammatory activities of the body.
In U.S. Pat. No. 6,548,556 it is reported that a proteolytic enzyme has in part or in total the capacity to hydrolyze peptide amide bonds and that such enzymes may also have some inherent lipolytic and/or amylolytic activity associated with the proteolytic activity, with the preferred proteolytic enzyme being papain. Other suitable proteolytic enzymes include trypsin, chymotrypsin, streptokinase, streptodormase, ficin, pepsin, carboxypeptidase, aminopeptidase, chymopapain, bromelain, as well as other suitable enzymes, such as pancreatin, trypsin, collagenase, keratinase, carboxylase, aminopeptidase, elastase, and aspergillopeptidase. Pancreatin contains a mixture of peptide hydrolases/proteases (trypsin, chymotrypsin, elastase, carboxypeptidase A, carboxypeptidase B), lipolytic enzymes (lipase, phospholipase A2, phospholipase B, cholinesterase, cholesterol esterase), glycosidases (α-amylase, glucosidase), and nucleases (deoxyribonuclease I, deoxyribonuclease II, ribonuclease).
Mixed debriding agent enzymes from Bacillus subtilis are described in U.S. Pat. No. 3,409,719. This enzyme product is reported to exhibit proteolytic activity against casein (phosphoprotein) and similar activity against hemoglobin (metalloprotein). It also exhibits amylolytic activity against gelatinized starch. It is capable of rapid lysis of fibrin, denatured collagen, elastin and exudate, which are reported to be the principal tissue protein components in the wound. The mixed enzyme compositions are shown in Table I of U.S. Pat. No. 3,409,719 to contain a minimum of amylase to proteases, ranging from 6.3% amylase and 93.7% proteases to 18.2% amylase and 81.8% proteases.
Severe burn wounds require surgical debridement in order to quickly apply antimicrobials and dressings to reduce the risk of infection, (e.g., Pseudomonas aeruginosa sepsis, U.S. Pat. No. 4,772,465) and to prepare the wound bed for healing or subsequent skin grafting. Because of the potential of enhanced bleeding from viable tissue removal, enzymatic debridement may be preferable for removal of necrotic tissue. Wound healing is also difficult for diabetics due to cardiovascular insufficiency and neuropathy; hence, enlarging the diabetic ulcer size by surgical debridement may not be desirable, and a critical need exists for improved debridement for diabetic ulcers.
In International Patent Publication Number WO1999046368 a method for treating wounds comprising the step of administering an effective amount of a carbohydrate-active enzyme is discussed, which is reported to have broad-specificity for debriding burns and other wounds. Because of the high concentrations of glycosaminoglycans (GAGs) in skin, in burn patients enzymes that degrade glycosaminoglycans are considered to be useful adjuncts to burn wound debridement. Glycosaminoglycans are sugar chains consisting of repeating polymers of acidic polysaccharides, composed of building blocks of the following sugars in various combinations: galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, galacturonic acid and iduronic acid. It is known that carbohydrates have important roles in the functioning of living organisms. In addition to their metabolic roles, carbohydrates are structural components of the human body, being covalently attached to numerous other entities such as proteins (i.e., as glycoproteins). Since human skin is reported to contain 10% by weight of glycosaminoglycans (which include heparin, heparan sulfate, chondroitin sulfate, hyaluronic acid (hyaluronan), dermatan sulfate, and keratan sulfate, with chondroitin sulfate being the most prevalent glycosaminoglycan. Chondroitin sulfate also has β-1,3- and β-1,4-linkages between predominant monomeric units. The term “carbohydrate-active enzyme” as used to specifically encompass carbohydrate reducing enzymes, where examples of such enzymes include glycosaminoglycan reducing enzymes such as hyaluronidases, chondroitinases, dermatanases, heparanases, hepanrnases and keratanases, with preferred carbohydrate-active enzymes of chondroitinases and hyaluronidases.
Infection control is also a significant unaddressed need in debridement. Burn eschar is typically dry necrotic tissue not readily infected, but sepsis can occur with second and third degree burns; therefore, having antimicrobials in the eschar and wound bed during early stages of eschar (necrotic tissue) removal is highly desirable. In contrast to burn eschar, chronic wound (diabetic ulcer) necrotic tissue appears to promote bacterial colonization, supported by the presence of water and nutrient sources from dead cells and debris. Necrotic tissue in wounds may be associated with infection, while a majority of chronic wounds are infected with microorganism biofilm.
A difficulty in the use of proteolytic enzymes for debridement of necrotic tissue is their ability for auto-digestion in aqueous solution, in addition to their potential difficulty with adverse allergic events, hypersensitivity and/or lack of efficacy. It is thus desirable to identify a debridement composition, having a high debridement efficacy for necrotic tissue, that is clinically simple to use, exhibits a suitable shelf-life, and is non-allergenic.